Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses

Sukal, Amit C.; Kidanemariam, Dawit B.; Dale, James L.; Harding, Robert M.; James, Anthony P.

doi:10.1016/j.virol.2019.01.013

Cited by 18 publications

(17 citation statements)

References 51 publications

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“…A badnavirus‐biased RCA approach (Sukal et al . , ) was used to amplify viral circular DNA. Briefly, a mixture of 32 degenerate badnavirus primers (Sukal et al .…”

Section: Methodsmentioning

confidence: 99%

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

“…A badnavirus-biased RCA approach (Sukal et al, 2019) was used to amplify viral circular DNA. Briefly, a mixture of 32 degenerate badnavirus primers (Sukal et al, 2019) at a final concentration of 0.4 μM of each primer, 1 × Φ29 buffer (NEB) and 1 μl (c. 500 ng) of TNA was made up to a final volume of 10 μl with sterile NF-H 2 O and denatured at 95 °C for 3 min, cooled to 4 °C and placed on ice. A 10 μl reaction mixture consisting of 2.5 μM exo-resistant random hexamers (Thermo Fisher Scientific), 1 × Φ29 buffer, 2 ng/μl bovine serum albumin (BSA), 4 mM DTT, 15 mM dNTPs, 5 U/μl of Φ29 DNA polymerase (Thermo Fisher Scientific) and sterile NF-H 2 O to make up the final volume, was prepared and added to each denatured sample.…”

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

“…RCA products were independently digested with EcoRI and SphI (NEB). These enzymes were selected following in silico restriction analysis of published yam badnavirus genome sequences and previously published work (Sukal et al, 2019). Digest products were electrophoresed through 1.5% agarose gels, stained with SYBR Safe (Thermo Fisher Scientific) and fragments of interest were excised, purified and cloned as described in Sukal et al (2017).…”

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

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Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

et al. 2020

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Yam is an important food staple in many Pacific Island countries, but its production is limited by several factors including virus infection. The presence of viruses also hinders the exploitation of the crop by restricting the international dissemination of yam germplasm due to quarantine concerns. In this study, a total of 224 yam accessions present in the Pacific germplasm collection at SPC‐CePaCT, Fiji, were tested for the presence of badnaviruses using a combination of rolling circle amplification (RCA) and sequencing. Of these, 35 tested positive for either Dioscorea bacilliform AL virus (DBALV) or Dioscorea bacilliform AL virus 2 (DBALV2). DBALV was only detected in samples from Vanuatu and Tonga, while DBALV2 was restricted to Papua New Guinean samples. Ten complete genome sequences of DBALV and DBALV2 were obtained and pairwise comparison of the partial reverse transcriptase/ribonuclease H (RT/RNase H)‐coding sequence revealed identities ranging from 89% to 100% and 92% to 100%, respectively. The results suggest that DBALV and DBALV2 are the prevalent badnavirus species infecting yams in the Pacific region.

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“…A badnavirus‐biased RCA approach (Sukal et al . , ) was used to amplify viral circular DNA. Briefly, a mixture of 32 degenerate badnavirus primers (Sukal et al .…”

Section: Methodsmentioning

confidence: 99%

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

Section: Rca Cloning and Sanger Sequencingmentioning

confidence: 99%

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Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

et al. 2020

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“…This amiRNA-based silencing strategy was applied effectively in many plants to combat plant viruses: cotton leaf curl Kokhran virus (CLCuKoV-Bu) [19], Cucumber mosaic virus (CMV) [20], CymMV and ORSV [21]. Serological method, ELISA, immunosorbent electron microscopy (ISEM) [22], IC-PCR [23], RT-PCR [24], rolling circle amplification (RCA) [25] are the most widely used reliable detection techniques for SCBV. New strategies are under development for improved diagnoses and cure of badnaviruses [26].…”

Section: Introductionmentioning

confidence: 99%

An Algorithmic framework for genome-wide identification of Sugarcane (Saccharum officinarumL.)-encoded microRNA targets against SCBV

Ashraf¹,

Feng

Ashraf

et al. 2020

Preprint

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Sugarcane Bacilliform Virus (SCBV) is considered an economically the most damaging pathogen for sugarcane production worldwide. Three ORFs are characterized in a single molecule of circular, ds-DNA genome of the SCBV, encoding for hypothetical protein (ORF1), DNA binding protein (ORF2) and Polyprotein (ORF3). The study was aimed to predict and comprehensively evaluate sugarcane miRNAs for the silencing of SCBV genome using in-silico algorithms. Computational methods were used for prediction of candidate miRNAs from sugarcane (S. officinarum L.) to silence the expression of SCBV genes through translational inhibition by mRNA cleavage. Mature sugarcane miRNAs were retrieved and were assessed to hybridization with the SCBV genome. A total of fourteen potential candidate miRNAs from sugarcane were computed by all the algorithms used for the silencing of SCBV. A consensus of three algorithms predicts hybridization sites of sof-miR159e at common locus 5534. The miRNA-mRNA interaction was estimated by computing free-energy of miRNA-mRNA duplex using RNAcofold algorithm. Regulatory network of predicted candidate miRNAs of sugarcane with SCBV ORFs, generated using Circos, identify novel targets. Consequently, detecting and discarding inefficient amiRNAs prior to cloning would help suppressed mutants faster. The efficacy of predicted candidate miRNAs was evaluated to test the survival rate of the in vitro amiRNA-mediated effective badnaviral silencing and resistance in sugarcane cultivars.

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Virus Diseases of Tropical Crops

Geering

2021

Encyclopedia of Life Sciences

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Virus diseases are serious constraints to the productivity and profitability of a wide range of tropical crops. Identification of the causal viruses and understanding their epidemiology is the key to estimating the incidence and economic impact of the diseases they cause, and to devising virus management strategies. Epidemics result from interactions between virus, host plant, vector and environmental factors, and every epidemic can be considered to be a unique pathosystem in which each of the components contributes to the epidemic, and in which none are limiting. Accurate pathogen diagnosis is the key to managing diseases. The main virus and viroid diseases of 18 different crops are listed and pathosystem descriptors provided for each based on the mode of spread, which in turn informs the different management strategies. The Tenth Report of the International Committee on Taxonomy of Viruses, which is available online at https://talk.ictvonline.org/ictv‐reports/ictv_online_report/ , is an essential reference for virologists working with tropical crops. Key Concepts Identification of viruses or viroids is the key to managing the diseases they cause. Koch's postulates are applied to establish causal associations between viruses/viroids and disease. Viruses/viroids are identified by their biological, morphological and genetic properties using classical and molecular methods. The universal database for viruses/viroids published by the International Committee on Taxonomy of Viruses is the resource for classification. Rapid diagnostic methods rely on nucleotide sequence comparisons with publicly available databases. Epidemics are driven by virus, vector, host, environment, time and human activity. The vector–virus interaction is the main determinant of the rate, range, timing and pattern of disease spread. Viruses/viroids of tropical plants have unique pathosystems. A full description of a pathosystem is required to sustainably manage virus epidemics. Management of epidemics can be achieved by destabilising or downregulating one or more components of the relevant pathosystem.

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Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses

Cited by 18 publications

References 51 publications

Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

An Algorithmic framework for genome-wide identification of Sugarcane (Saccharum officinarumL.)-encoded microRNA targets against SCBV

Virus Diseases of Tropical Crops

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